Natural endonucleases have the ability to distinguish and cleave a given DNA sequence with remarkable selectivity, but this level of finesse has not yet been achieved with synthetic small molecule analogs (Truwick et al., 1998; Ott et al., 1999). It is of great interest to generate an artificial endonuclease to target sequences of choice, not only for their biochemical utility, but for the pharmaceutical impact of cleavage agents which could target a single sequence in the genome. Such an agent could serve to regulate transcription of given genes, such as ras, P16, lac or P53 which often go awry in cancers (Fahraeus et al., 1999; Roth et al., 1997), or to down-regulate gene expression for the treatment of genetic disease, inflammation, or infection.
A number of different approaches have been used to prepare artificial nucleases having sequence specificity. One approach is to link a nucleic acid binding domain isolated from one protein to a domain from another protein that exhibits nuclease activity. For example, the FokI endonuclease DNA binding domain (Li et al., 1992; Li et al., 1993) has been fused to the Drosophila Ubx homeodomain, to zinc-finger DNA binding domains, and to the yeast Gal4 DNA binding domain (Kim et al., 1994: Kim et al., 1996; Huang et al., 1996; Kim et al., 1998). Another approach is to link an oligonucleotide that binds to a specific nucleotide sequence in a target nucleic acid with the domain of a protein that has nuclease activity. Examples of protein domains that have been used with this type of approach include staphylococcal nuclease, RNase S, and E. coli RNase H (Zucherman et al., 1989; Zucherman et al., 1988; Kanaya et al., 1992; Uchiyama et al., 1994).
Metal and organic complexes that cleave nucleic acid have also been linked to oligonucleotides. Examples of metal and organic complexes that have been used to prepare this type of artificial nuclease include a terpyridine group, a lanthanide-complexing iminodiacetate residue, Eu(III) chelated by a pentadentate texaphyrin ligand, imidazole, and histamine groups (Bashkin et al., 1994; Matsumura et al., 1994; Magda et al., 1994; Reynolds et al., 1996; Vlassov et al., 1997).
Basile et al. (1987) report that plasmid DNA was incubated with chelate complexes of non-redox-active metal ions Zn(II) and Cd(II) (e.g., with phenanthroline) to produce nicked DNA. More recently, hydrolysis of supercoiled DNA has also been achieved with Ln(III) complexes (Tanaka et al., 1990; Hayashi et al., 1993; Rammo et al., 1996; Rammo et al., 1996a; Zhu et al., 1998), a dinuclear Fe(III) complex (Schnaith et al., 1994), Co(III) complexes (Dixon et al., 1996; Hettich et al., 1997a), Cu(II) complexes (Hegg et al., 1996; Itoh et al., 1997), and various divalent transition metal ions (Sagripanti et al., 1989; Hashimoto et al., 1996; Rammo et al., 1996b). A dinuclear Co(III) complex was among the most efficient complexes (Hettich et al., 1997b).
Hydrolysis of linear DNA has been achieved with heterogeneous Ce(IV) hydroxide systems which also form in situ from Ce(III) salts and dioxygen (Komiyama et al., 1993a; Tagasaki et al., 1994; Komiyama et al., 1994a, 1994b, 1994c). Redox-active Cu(II) phenanthroline complexes were also able to produce nicked DNA from a supercoiled template. However, in this case T4 DNA ligase was not able to religate the nicked DNA, suggesting that the Cu(II) complexes utilized a mechanism of cleavage other than that used by the other metals described above, i.e., not via hydrolysis.
DNA binding domains are generally small and compact, and so in some instances out of the context of a full length DNA binding polypeptide, the domain may not make a sufficient number of contacts with DNA to specify both a unique target site and bind with reasonable affinity. To overcome this problem, arms or tails that recognize additional features of the DNA have been added to DNA binding domains (Kissinger et al., 1990; Wolberg et al., 1991), or the polypeptide has been engineered to form either homo- or heterodimers (Schwabe et al., 1993; Glover & Harrison, 1995; Schwage et al., 1994; Klemm et al., 1994). One example is the artificial protein ZFHD1 that was constructed by linking two zinc-fingers with a homeodomain (Pomerantz et al., 1995).
Thus, there is a continuing need for an artificial endonuclease that has specificity for a particular nucleic acid sequence.